Design, synthesis and in-vitro evaluation of fluorinated triazoles as multi-target directed ligands for Alzheimer disease

Article abstract of DOI:10.1016/j.bmcl.2021.127999

Alzheimer disease is multi-factorial and inflammation plays a major role in the disease progression and severity. Metals and reactive oxygen species (ROS) are the key mediators for inflammatory conditions associated with Alzheimer's. Along multi-factorial nature, major challenge for developing new drug is the ability of the molecule to cross blood brain barrier (BBB). We have designed and synthesized multi-target directed hexafluorocarbinol containing triazoles to inhibit Amyloid β aggregation and simultaneously chelate the excess metals present in the extracellular space and scavenge the ROS thus reduce the inflammatory condition. From the screened compound library, compound 1c found to be potent and safe. It has demonstrated inhibition of Amyloid β aggregation (IC₅₀ of 4.6 μM) through selective binding with Amyloid β at the nucleation site (evidenced from the molecular docking). It also chelate metals (Cu⁺², Zn⁺² and Fe⁺³) and scavenges ROS significantly. Due to the presence of hexafluorocarbinol moiety in the molecule it may assist to permeate BBB and improve the pharmacokinetic properties. The in-vitro results of compound 1c indicate the promiscuity for the development of hexafluorocarbinol containing triazoles amide scaffold as multi-target directed therapy against Alzheimer disease.

Full text of DOI:10.1016/j.bmcl.2021.127999

Bioorg. Med. Chem. Lett. 42 (2021) 127999
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and in-vitro evaluation of fluorinated triazoles as
multi-target directed ligands for Alzheimer disease
,
,
,
Dalvi Tanaya d, Dewangan Bhaskara d, Agarwal Gopalb d, Shinde Suchita Dattatraya,
Jain Alokb e, Srivastava Akshayc, Sahu Bichismitaa
,
,*
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, Opposite Air Force Station, Palaj,
Gandhinagar, Gujarat 382355, India
b Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, Opposite Air Force Station, Palaj, Gandhinagar,
Gujarat 382355, India
c Department of Medical Devices, National Institute of Pharmaceutical Education and Research(NIPER) Ahmedabad, Opposite Air Force Station, Palaj, Gandhinagar,
Gujarat 382355, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Alzheimer disease is multi-factorial and inflammation plays a major role in the disease progression and severity.
Metals and reactive oxygen species (ROS) are the key mediators for inflammatory conditions associated with
Alzheimer’s. Along multi-factorial nature, major challenge for developing new drug is the ability of the molecule
to cross blood brain barrier (BBB). We have designed and synthesized multi-target directed hexafluorocarbinol
containing triazoles to inhibit Amyloid β aggregation and simultaneously chelate the excess metals present in the
extracellular space and scavenge the ROS thus reduce the inflammatory condition. From the screened compound
library, compound 1c found to be potent and safe. It has demonstrated inhibition of Amyloid β aggregation (IC50
Alzheimer disease
Multi-target directed ligands
Fluorinated triazole-amides/amines
Metal Chelation
ROS scavanging
of 4.6 μM) through selective binding with Amyloid β at the nucleation site (evidenced from the molecular
docking). It also chelate metals (Cu+2, Zn+2 and Fe+3) and scavenges ROS significantly. Due to the presence of
hexafluorocarbinol moiety in the molecule it may assist to permeate BBB and improve the pharmacokinetic
properties. The in-vitro results of compound 1c indicate the promiscuity for the development of hexa-
fluorocarbinol containing triazoles amide scaffold as multi-target directed therapy against Alzheimer disease.
Alzheimer’s disease (AD) is a progressive neurodegenerative disor-
der which involves the degeneration of cholinergic neurons and one of
the major causes of dementia in geriatric population.1 Current treatment
of AD restricted to the symptomatic treatments by anticholinesterases
(Rivastigmine, Donepezil, Galantamine) and NMDA receptor antago-
nists Memantine.2 With growing rate of attrition of drugs in advance
stage of clinical trials3 and limited therapy urged the researchers to
discover new drugs towards AD. Histological analysis of the brain dis-
sects of AD patient established the presence of extracellular deposit of
peptide Amyloid β plaque,4 and intracellular neurofibrillary tangle of
hyper phosphorylated tau protein.5 Proteopathy in AD is enhanced by
other factors such as oxidative stress and inflammation and metal dys-
homeostasis.6 In normal brain, 0.04 mg/g fresh tissue sample contains
iron with concentration of ~ 720 µM and abundantly found in basal
ganglia region. Zinc concentration is 10 times higher in brain as
compared to the serum and estimated to be around 150 µM, where as the
concentration of copper in human frontal lobe and cerebellum is in the
range of 60 ~ 110 μM.
As compared to healthy parenchyma in brain, these metals are found
5.7, 2.8, and 3.1 times higher (copper, zinc and iron respectively) and
aggravates the aggregation of Amyloid β through binding at N-terminus
His6, His13 and His14 residue of the peptide.7 Metals such as Cu (I/II)
and Fe (II/III) also produce reactive oxygen species (ROS) which leads to
neuronal cell death.8,9 Clioquinol an analogue of chloroquine, has
shown moderate affinity towards metal which shift the entrapped metal
towards essential metallo-cycle in brain and currently under phase II
clinical trial.10
Complex disease which involves multiple pathways needs the
* Corresponding author.
E-mail address: bichismita77@gmail.com (B. Sahu).
d
Equal contribution.
e
Current affiliation: Department of Bio-Engineering, Birla Institute of Technology, Mesra, Ranchi, India.
https://doi.org/10.1016/j.bmcl.2021.127999
Received 4 January 2021; Received in revised form 15 March 2021; Accepted 19 March 2021
Available online 9 April 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

T. Dalvi et al.
Bioorganic & Medicinal Chemistry Letters 42 (2021) 127999
diseases with anti-inflammatory activity.19 Triazole based compounds
are now under study for the treatment of many neurological disorders
such as epilepsy. Di-triazole based compounds have been explored
recently as therapeutics for the treatment of Alzheimer’s disease.20
Recently, triazole based compound QTC-4-MeOBnE was selected
through virtual screening and shown promising results in-vivo.21 We
have selected triazole as part of the scaffold in aniticipation that it may
exert anti-inflammatory/anti-oxidant activity and simultaneously can
be explored for the metal chelation due to the presence of heteroatom.
Therefore, by placing suitable functional group adjacent to 1,2,3 triazole
moiety can lead to effective metal chelation. Therefore, triazole amides
and triazole methylamines are incorporated as linker, which can hold
the two aromatic rings at optimum distance for Aβ interaction and
simultaneously chelate the metal and demonstrate anti-oxidant/anti-
inflammatory properties (Fig. 1).
Fig. 1. Designing of the scaffold to target multiple pathways related to AD.
therapeutic strategy which can engage multiple signalling pathways
simultaneously. Multi-target directed ligand (MTDL) is a strategy in
drug discovery process, where the single molecule is designed with its
multiple pharmacophoric properties to engage multiple targets simul-
taneously.11 There is a substantial portion of the approved drugs are
multi-target-directed drugs for other diseases also.12
Hexafluorocarbinol group is an interesting pharmacophore with
hydroxyl group flanked by two CF3 group and has been explored pre-
viously for Aβ inhibition. HFIP (hexaflouroisopropanol) is used as sol-
vent for disaggregating the Aβ peptide and molecules containing this
functional moiety are found as bioactive. Therefore, incorporation of
hexafluorocarbinol in the molecule may increase the disaggregation
properties of molecule and due to the presence of multiple fluorines it
may increase the cellular permeability and plasma half-life of the drug.
We have designed two series of compounds (series A, containing
triazole amide and series B containing triazole amine) for further syn-
thesis and in-vitro evaluations
Amyloid aggregation is the major hallmark for AD and the aggre-
gation process is induced by many biological factors. The hydrophobic
core with sequence KLVFF undergoes self aggregation and produce toxic
oligomers and plaues.13 Therefore, controlling amyloid aggregation
through those pathways can be effective. Mitochondrial dysfunction and
oxidative stress are key players in the pathogenesis of Alzheimer’s dis-
ease.14 Aβ species can also induce oxidative stress, by entering into
mitochondria and increasing the production of reactive oxygen species
(ROS), which, in turn, damage important macromolecules.15 The Zn2+
and Cu2+ ions can bind to Aβ and induce further formation of amyloid
aggregates.16,17 Based on the disease molecular mechanism of action, it
is postulated molecules which can inhibit Aβ aggregation by blocking
the hydrophobic core and exhibit antioxidant/anti-inflammatory prop-
erties with metal chelation ability can be a potential therapeutic mole-
cule to treat AD.
Molecular docking was performed prior to the synthesis of com-
pounds. The compounds were docked against Aβ42 Monomer (PDB ID-
1IYT) using Autodock 4.2 software as discussed in detail in the meth-
odology section. The purpose of docking was to investigate whether the
compounds acquire the best possible interaction with the monomer
particularly with the hydrophobic core bearing the KLVFF sequence (AA
16–20) which is considered to be the self-recognition site. During the
nucleation phase, Aβ42 monomers aggregate to form the oligomers and
then form fibrils via this self-recognition site.
The driving force for aggregation of Amyloid beta (Aβ) peptide is the
core residue penta-peptide fragment (KLVFF). Moreover, the bis-
phenylalanine (FF) dipeptide sequence undergoes self-aggregation to
form diverse nano-structures which is now currently utilized in
designing novel nano material.18 Many aromatic compounds including
curcumin have found to inhibit Aβ aggregation efficiently and they have
common feature of two aromatic rings separated by an optimal linker.
Therefore, we have incorporated two aromatic residues in our scaffold,
anticipating that aromatic rings may interact with the phenyl alanine
We have performed docking studies with representative compounds
from both series (1a from Series A and 2a from Series B). Four different
grids are generated and compounds are docked (Table 1).
Representative examples from both the series have demonstrated
similar binding affinity with slightly preferential activity towards grid 2
which contain the hydrophobic core fragment KLVFF.
A closer analysis of interaction pattern of 1a and 2a suggested that,
the hexafluorocarbinol group present in the compound, exhibits certain
specific interactions with the amino acids of the peptide and anchors the
compound in such a way that the phenyl ring of the scaffold which in-
teracts with the aggregation prone diphenylalanine FF (Phe19 and 20)
motif. Hexafluorocarbinol group plays an important role since without
this group, although molecule binds with Aβ, but the aggregation prone
diphenyl alanine (FF) remains exposed (Fig. 2). These phenylalanine
residues are shown to play a predominant role in the fibril formation and
aggregation process through pi-pi stacking type of interactions.22,23
These residues when blocked, interactions of one monomer with the
other may thus be prevented which will in turn hinder the Aβ aggre-
gation process.
residue of KLVFF fragment of Aβ peptide through π-π-interaction and
block the aggregation.
Triazoles as potential pharmacophore for the neurodegenerative
Table 1
Binding energy of compound1a and 2a.
Furthermore, other favorable interactions such as electrostatic,
hydrogen bonding and Van der Waals types of interactions are observed.
The difference or change in the intermolecular interactions and
binding energy was evaluated with the positional switch of the hexa-
fluorocarbinol (HFC) group. Similar pattern of interactions is observed
for the compound 1g (Fig. 4).
Compound
1a
Grid
Binding Energy
(Kcal/mol)
1
2
3
4
1
2
3
4
ꢀ 4.12
ꢀ 4.73
ꢀ 4.58
ꢀ 3.41
ꢀ 4.29
ꢀ 4.69
ꢀ 3.84
ꢀ 3.61
In case of 1g, the phenyl group attached to amide was found to be
engaged with Phe19 instead of the phenyl attached to triazole. It also
retained all the conventional interactions as 1a.
2a
When amide was replaced with methylamine, compound 2a has
demonstrated similar interaction with preferential binding at grid 2 and
retained favorable interaction with Phe19 (supporting information,
Grid 1: AA 1–10; Grid 2: AA 11–21; Grid 3 AA 22–31; Grid 4 AA 32–42.
2

T. Dalvi et al.
Bioorganic & Medicinal Chemistry Letters 42 (2021) 127999
150
100
50
150
100
**
*
**
***
***
***
***
***
***
***
***
***
***
50
0
***
***
***
***
***
0
Fig. 2. Process of Amyloid β (Aβ) aggregation. The Aβ monomer aggregates to
form oligomer which further aggregates to form fibrils which are highly
neuro-toxic.
Fig. 5. Cytotoxicity Evaluation: The cytotoxicity of the synthesized compounds
(1a-1j and 2a-2j) were evaluated using PC-12 cells. The cells were incubated
with the compounds at 40 μM concentrations for 24 h and then the cell viability
was evaluated using Alamar Blue reagent. The percentage cell viability after
treatment was compared with cells that were not treated.
the coupling of corresponding triazole acids (3) and the aryl amine (4)
by using standard coupling reagents such as DCC and HOBt. Triazole
acids were obtained from the basic hydrolysis of corresponding triazole
esters (5). Triazole esters were in turn prepared via click reaction of aryl
azides (6) and ethyl propiolate (7). All the synthesized compounds (1 a-
k) were isolated in moderate to good yield.
Table 2
Synthetic scheme and structures of the compounds from series A &B.
Fig. 3. The intermolecular interactions and binding affinities of (a) Triazole-
amide (without Hexafluorocarbinol) and (b) Compound 1a with Aβ42 mono-
mer have been depicted. In both the images, the 3D representation is displayed
on the left and the 2D representation on the right, whilst the colour code for
each interaction being displayed at the bottom. The Aβ42 monomer is depicted
in ribbon form in red colour while the designed molecules are represented in
ball and stick. Interacting residues of Aβ42 monomer were displayed in stick
representation. Colour key of the atoms represented is as follows: Carbon-Grey,
Hydrogen- White, Oxygen- Red, Nitrogen- Blue, and Fluorine- Cyan.
Compound
1a
Structure
Compound
2a
Structure
1b
1c
2b
2c
1d
1e
2d
2e
1f
2f
Fig. 4. Intermolecular interactions and binding affinity of Compound 1 g with
Aβ42 monomer. The positional switch of hexafluorocarbinol group does not
predominantly affect the anchoring tendency of the molecule and thus results
1g
1h
2g
2h
into similar π-π interaction as observed with Compound 1a (Fig. 3). The colour
coding for the atoms is the same as represented in Fig. 3.
Fig. S2).
Therefore, compound library is generated where; phenyl rings have
been substituted with electron donating as well as withdrawing groups.
To our delight, we have found similar trend in the in-vitro Aβ42 aggre-
gation inhibition study (Fig. 5) of triazole amides 1c and 1j (positional
switch of hexafluorocarbinol group).
1i
1j
2i
2j
Compounds were synthesized by using known synthetic protocols
(Scheme 1).24 For series A, triazole amides (1 a-k) were prepared from
3

T. Dalvi et al.
Bioorganic & Medicinal Chemistry Letters 42 (2021) 127999
Similarly, series B triazole amines were synthesized via one pot re-
action of aryl azide (6), propargyl bromide (8) and corresponding aryl
amines (4) in water in the presence of copper iodide, triethylamine and
surfactant. Although, a step-wise synthesis was tried by utilizing the
click reaction of arylazide with propargyl bromide (9) followed by
condensation with arylamine in the presence of base, we have failed to
isolate the intermediate. One pot reaction of arylamine, aryl azide and
propargyl bromide in water in the presence of catalyst copper iodide and
base triethylamine also failed to yield any desired product.25 Surfactants
are known to enhance the reaction rate, therefore we have screened
surfactants (SDS and DOSS) in water for above mentioned reaction.
Reactions were preceded well and all triazole amines (2a-i) were iso-
lated in moderate yields. Synthesized compounds are summarized in
1
Table 2. Compounds were characterized by H, 13C NMR and HRMS.
Presences of fluorine were confirmed by 19F NMR.
Prior to performing Aβ binding assay, we have screened the com-
pounds for their cytotoxicity against neuronal cell (PC-12) since, the
main limitation for the lead compounds in the discovery and develop-
ment process is their intolerable toxicity profile. The toxic effects of the
synthesized compounds were evaluated against PC12 neuronal cell line
at a single concentration of 40 µM using Alamar Blue reagent. The re-
sults (Fig. 5) indicate that a fair number of compounds posed cell
viability >60% in PC12 cells at the employed concentrations.
Fig. 7. Copper Chelating Effect: The compounds were incubated with Aβ42 (10
µM) with 10 µM Cu+2 concentration for 24 h (at 37 ◦C, 180 rpm) and the
percentage of Aβ42 fibrillation formation was calculated using Thioflavin T
reagent. The statistical significance was evaluated using Dunnet test, and
compared with Aβ42 kept for fibrillation for 24 h. (n = 3, *** vs Aβ42 alone,
### means vs Aβ42 + Cu+2).
We observed that when hexafluorocarbinol is incorporated at the
right-hand aromatic ring compound (1g) was found to be less toxic as
compared to the left-handed hexafluourocarbinol substituted counter-
part (1a). Left-handed hexafluorocarbinol substituted compounds with
polar group substitutions (OMe, Cl, Br) in right-hand aromatic ring or
replacement of aromatic ring with heteroaromatic ring were cytotoxic
for triazole amides. Only 3, 4 dimethyl substitutions on either of the
aromatic ring (1j, 1c) were found to be safer for further in-vitro evalu-
ation and cytotoxicity did not altered much by positional switch of
hexafluorocarbinol group. Switching the linker from triazole amide (1a)
to triazole methylamine (2a), found to be toxic. Substitutions such as 3,4
dimethoxy, 3,4 dimethyl and 3,4, 5 trimethoxy in left-hand aromatic
ring found to possess good cell viability as compared to halo sub-
stitutions. However, unlike the 3,4 dimethyl substituted amides (1j and
1c), positional switch of hexafluorocarbinol groups from left hand aro-
matic ring to right hand resulted considerable cytotoxicity.
and inhibit aggregation following standard Thioflavin T assay protocol.
We have selected compound 1 g, 1c and 1j from triazole amides to screen
for their ability to inhibit Aβ42 aggregation. Compound 1g failed to
inhibit Aβ42 aggregation. However, dimethyl substitution at either aro-
matic ring (1c and 1j) leads to effective inhibition of aggregation at the
concentration of 40 μM. The inhibition was comparable to that of cur-
cumin at the (Fig. 6). Compounds with 3,4 dimethyl substitution (2c)
and 3,4 dimethoxy substitution (2b) at one of the aryl group were
selected from the triazole methylamine scaffold. To our surprise, com-
pound with 3,4 dimethyl substitution (2c) failed to inhibit aggregation
(Fig. 6). However, dimethoxy substituted triazole amine (2b) demon-
strated similar inhibition as of compound 1c.
Compounds which have demonstrated good cell viability (less toxic)
were selected and screened for their ability to bind with Aβ monomer
Presence of metals such as Zn, Cu and Fe are known to accelerate the
aggregation rate of Aβ. Therefore, to find a potent compound which can
150
100
50
***
***
***
***
0
Fig. 6. Amyloid beta fibrillation formation: The compounds were incubated
with Aβ42 (10 µM) at 40 µM concentration and for 24 h (at 37 ◦C) and the
percentage of Aβ42 fibrillation formation was calculated using Thioflavin T
reagent. The statistical significance was evaluated using Dunnet test, and
compared with amyloid beta 42 kept for fibrillation for 24 h. (*** means n = 3,
p < 0.05).
Fig. 8. Metal chelation of compound 1c; The UV–Vis spectra of compound 1c
incubated with Cu+2, Zn+2 and Fe+3
.
4

T. Dalvi et al.
Bioorganic & Medicinal Chemistry Letters 42 (2021) 127999
1.4
1.2
1.0
0.8
0.6
0.4
0.2
y= 0.19X+0.022
R2= 0.93
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
1
2
3
4
5
6
Ratio of 1c: Copper
1c
1c: Cu (1:1)
1c: Cu (1:2)
1c: Cu (1:3)
1c: Cu (1:4)
1c: Cu (1:5)
1c: Cu (1:6)
Fig. 11. ROS scavenging effect of 1c compound: compound 1c ROS scavenging
effect was analysed using DCFDA analysis, after LPS inflamed PC-12 cells
treatment with 1c compound (40 µM) after 24 h a) FACS analysis dot blot
showing cell population percentage (ROS (low) vs ROS (High)) b) Bar graph of
FACS dot blot ROS level after 1c compound treatment (n = 3, *** p < 0.001).
240
260
280
300
320
Wavelength (nm)
Fig. 9. UV–vis spectra of compound 1c at different ratio of Cu+2 in HEPES
buffer at room temperature.
demonstrated absorption maximum around 255 nm. When incubated
with the compound in 1:1 ratio with CuSO4, Zn SO4 and FeCl3 at 50 µM
concentration for 30 min (in HEPES buffer at pH 7.4, at room temper-
ature) a significant hyperchromic shift observed for all the metals. The
change in the intensity of the sample in the presence of metal ion con-
firms the interaction of the compound. To confirm further, we have
titrated the compound 1c with copper salts of varying ratio (1c: M = 1:0,
1: 1, 1:2, 1:3. 1:4. 1:5 and 1:6). The absorbance of the compound
increased in proportion of the salt which confirms the binding (Fig. 9).
When the binding study was performed with Zn+2 and Fe+3, increase
in absorbance obtained for Fe+3 with increment of concentrations was
observed as in case of Cu+2 (Fig. 10). However, minimal effects for in-
crease in concentration observed for Zn+2 (data not shown).
inhibit Aβ42 aggregation, we have decided to screen the hit compounds
from both series (1c and 2b) and tested for their ability to inhibit Aβ
aggregation in the presence of metal such as copper (Fig. 7).
Amount of aggregation found to be more in the presence of copper
without any compounds (Fig. 7). Compounds from both series were
found to reduce the aggregation up to 50% in the presence of copper. To
our observation, triazole amide 1c was found to inhibit aggregation
slightly better than the triazole amine 2b. Although, 1c and 2b are found
to be equipotent, amides are metabolically stable than amines. There-
fore, we have anticipated that 1c can be consider as scaffold of choice
and potent molecules can be further generated by doing systematic
structure–activity-relationship (SAR) study. Therefore, compound 1c
was envisaged as the potent hit molecule from the present screenings
and the IC50 for Aβ42 fibril formation inhibition was determined to be
Reactive oxygen species (ROS) involved in initiation and progression
of many inflammatory path-way. In AD, metal mediated redox reactions
produce many ROS which may contribute to neuronal cell damage and
death. Apart from metal chelation, 1,2,3 triazoles are privileged het-
erocyclic core with many interesting biological activities. Their stability
towards acidic/basic hydrolysis, high dipole moment (~5 D), ability of
the moiety to interact with biological target through hydrogen bonding,
ˆ
4.6 AµM (graph presented in supporting info as Fig. S61).
Metals play very important role in pathophysiology of AD and the
interaction of Aβ and biological relevant metals such as copper, zinc,
iron has already proven the formation of highly toxic aggregated com-
plex from Aβ. Therefore, many compounds with metal chelating ability
such as Desferroxamine (DFO), DP-109, Clioquinol etc., have been
evaluated and they have demonstrated success in animal model (Fig. 8).
Therefore, we have evaluated the hit compound 1c for its ability to
chelate metals such as copper, zinc and iron. Compound 1c
dipole–dipole interaction, π-π stacking make them very attractive for
medicinal chemist to design new scaffolds. Compounds possessing these
scaffolds are known to exhibit anti-inflammatory properties through
inhibition of ROS production. Therefore, we have investigated the effi-
cacy of the hit molecule 1c to scavenge ROS production in cell.
Inflammation was induced in PC12 cell through LPS (liposaccharide)
and cells are incubated in the presence of compound 1c at the concen-
Fe+3 Alone
Compound 1c Alone
(1:1) Compound 1c : Fe+3
1.0
0.8
0.6
tration of 40 μM as explained in methodology section. The efficiency to
scavenge ROS was evaluated using FACS (Fig. 11). The analysis was
done using DCFDA reagent. Initially, desired PC-12 cells population was
selected by applying gate in FSC area vs SSC area graph in unstained cell
populations. Thereafter, only singlet cells population was selected by
plotting and gating desired population in FSC area vs FSC height pop-
ulation. Propidium iodide (PI) staining was used to eliminate dead cells
population from the FACS study, and live cells were taken in to the
account for ROS activity. The DCFDA positive live cells population was
further evaluated plotting a dot blot (FL1 area vs FSC area) and applying
a quadrant to the population. The cell population shown in Fig. 6a
demonstrate ROShigh demonstrating the high fluorescence in PC-12 cells
due to DCFDA. The fluorescence intensity of DCFDA is directly pro-
portional to the ROS level, and hence can be used to distinguish ROS
activity in the cells. The percentage of DCFDA ROShigh population in LPS
(10 µg/mL) treated was much higher as compare to the PC-12 cells
treated with 1c compound (40 µM). This shows that there is a significant
reduction in ROS levels after incubating the cells with 1c compound
(1:3) Compound 1c : Fe+3
0.4
(1:5) Compound 1c : Fe+3
0.2
0.0
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
250
300
350
Wavelength (nm)
Fig. 10. UV–vis spectra of compound 1c at different ratio of Fe+3 in HEPES
buffer at room temperature.
5

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Bioorganic & Medicinal Chemistry Letters 42 (2021) 127999
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8
9
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chelation and inflammation. Compound 1c found to effectively inhibit
Aβ aggregation even in the presence of copper. It also demonstrated its
ability to chelate the metal and possess good anti-oxidant activity. Due
to the presence of multiple fluorine our hit molecule 1c, we speculate
that it may cross the BBB. Therefore, the designed scaffold (triazole
amide) may serve as the scaffold of choice for further SAR development
which may provide a potent multi-target directed ligand based drug
molecule for the treatment of AD.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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Authors are thankful to Prof. Kiran Kalia, Director, National Institute
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Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers,
Govt. of India for giving support as student fellowships and required
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